AGARICUS SILVATICUS - PROMISING FUNCTIONAL FOOD

Anita Klaus1, Maja Kozarski1, Jovana Vunduk1, Željko Žižak2, Miomir Nikšić1

1Institute for Food Technology and Biochemistry, Faculty of Agriculture, University of Belgrade, Nemanjina 6,

11080 Belgrade, Serbia 2Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000, Belgrade, Serbia

Agaricus silvaticus Schaeffer is often found in groups in mixed woodland and under trees in parks.

Highly efficient biological properties of this common, edible mushroom.

Important source of nutrients and nutraceuticals

Exhibits high antioxidant power

To determine their cytotoxic effect on malignant human breast cancer MDA-MB-453, cervical adenocarcinoma HeLa and myelogenous leukemia K562 cells.

O B J E C T I V E S

To determine the antibacterial ability of crude hot water extract (SV) and hot alkali extract (SNa) against selected foodborne Gram-positive and Gram-negative pathogenic bacteria by microdilution assay

To evaluate their antioxidant ability by measuring DPPH free radical scavenging activity, inhibition of lipid peroxidation, reducing power and chelating ability

hot water extraction

alcohol precipitation (supernatant)

hot alkali extraction (filter cake)

alcohol precipitation (supernatant)

hot alkali extract (SNa)

hot water extract (SV)

Determination of the MIC by broth microdilution method

Concentrations of mushroom extracts ranged from 20.0 to 0.0097 mg/mL.

Working concentrations of approximately 105-106 cfu/mL were used for antibacterial activity assay.

To indicate cellular respiration, 2,3,5-triphenyl tetrazolium chloride TTC (0.05%) was added to the culture medium.

The MIC was defined as the lowest sample concentration that exhibited complete inhibition of bacterial growth.

Quantitative determination of MIC, based on the color change caused by the enzymatic activity of viable microorganisms

hot water (crude) extract (SV) hot alkali extract (SNa)

G+ bacterial strains G- bacterial strains

Staphylococcus aureus ATCC 25923

Enterococcus faecalis ATCC 29212

Bacillus cereus ATCC 10876

Listeria monocytogenes ATCC 19115

Escherichia coli ATCC 25922

Salmonella enteritidis ATCC 13076

Shigella sonnei ATCC 29930

Yersinia enterocolitica ATCC 27729

Escherichia coli (0157:H7) ATCC 12900

MIC (mg/mL)

Bacterial strain Source SV SNa

Staphylococcus aureus ATCC 25923 5.0 ± 0.0a*1 2.5 ± 0.0b

Enterococcus faecalis ATCC 29212 5.0 ± 0.0a 0.3129 ± 0.0000b

Bacillus cereus ATCC 10876 5.0 ± 0.0a 1.25 ± 0.00b

Listeria monocytogenes ATCC 19115 5.0 ± 0.0a 2.5 ± 0.0b

Escherichia coli ATCC 25922 5.0 ± 0.0a 5.0 ± 0.0a

Salmonella enteritidis ATCC 13076 10.0 ± 0.0a 5.0 ± 0.0b

Shigella sonnei ATCC 29930 5.0 ± 0.0a 2.5 ± 0.0b

Yersinia enterocolitica ATCC 27729 10.0 ± 0.0a 5.0 ± 0.0b

Escherichia coli (0157:H7) ATCC 12900 5.0 ± 0.0a 5.0 ± 0.0a

Minimal inhibitory concentrations of tested mushroom extracts

*Data are expressed as mean ± standard deviation (n=3)

1Within the same row, means followed by different letters are significantly different at α=0.05 (ANOVA, Tukey`s HSD Test)

Both extracts, SV and SNa, inhibited the growth of all tested Gram-positive and Gram-negative bacteria; In most cases SNa possessed higher activity than SV (MIC - 0.3129 ± 0.000 - 5.0 ± 0.0 mg/mL and 5.0 ± 0.0 - 10.0 ± 0.0 mg/mL, respectively); The highest antibacterial potential of SNa was achieved against E. faecalis (MIC - 0.3129 ± 0.0000 mg/mL)

Evaluation of the antioxidant activity

The reducing power of SV and SNa was 1.4 ± 0.8 and 2.2 ± 1.6 at 5 mg/mL.

The inhibition of lipid peroxidation was determined by the conjugated diene method

At 10 mg/mL scavenging ability of SV toward DPPH radicals increased to 76.8 ± 1.2%, while 74.8 ± 2.3% was achieved in the presence of four times lower concentration (2.5 mg/mL) of SNa.

Chelating ability on ferrous ions

At particularly low concentration of 0.1 mg/mL SV and SNa inhibited peroxidation of 62.7 ± 3.1 and even 81.8 ± 1.9% of lipids.

DPPH free radical scavenging activity assay

Ferric-reducing antioxidant power assay

At 5 mg/mL SV and SNa chelated 87.7 ± 2.7% and 81.8 ± 1.4% of ferrous ions.

EC50a (mg extract / mL)

SV SNa

Inhibition of lipid peroxidation 0.25 ± 0.0 < 0.1

Reducing power 1.5 ± 0.1 Aa 0.72 ± 0.2 B

Scavenging ability on DPPH radicals 2.22 ± 0.2 A 0.26 ± 0.1 B

Chelating ability on ferrous ions 0.33 ± 0.0 A 0.09 ± 0.0 B

Extraction in hot sodium hydroxide solution contributed to the better antioxidant activity.

a Each value is expressed as mean ± standard deviation (n = 3). Within the same row, means followed by different letters are significantly different at α=0.05 (ANOVA, Tukey`s HSD Test)

An increase in antioxidant activity with increasing concentration of the extracts was confirmed in all applied assays.

SNa was better antioxidant shown by the lower EC50 values of inhibition of lipid peroxidation, ferric-reducing antioxidant power, DPPH scavenging ability, and ferrous ion-chelating ability.

Many wild mushroom could be a valuable natural source of antioxidant compounds, suggesting their potential role to be used in functional foods

In vitro cytotoxic activity

o human cervix adenocarcinoma HeLa o human myelogenous leukemia K562 o human breast carcinoma MDA-MB-453 cells

Cell survival was determined by MTT test

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Both investigated extracts exerted selective dose-dependent cytotoxic actions on malignant cells.

The decrease in survival of target cancer cells induced by the extracts is shown in graphs

IC50 [mg/mL] ± SD

extract HeLa K562 MDA-MB-453

SV 0.8 ± 0.1 1.3 ± 0.5 1.7 ± 0.5

SNa 0.7 ± 0.1 1.4 ± 0.2 1.6 ± 0.4

With IC50 values (concentration of extract that is required for 50% inhibition in vitro) ranging from 0.7 to 1.7 mg/mL, following continuous incubation, both examined extracts possess moderate cytotoxicity.

The highest cytotoxicity was found in a SNa treated HeLa cells (IC50=0.7 ± 0.1 mg/mL).

Crude hot water extract and hot alkali extract obtained from A. silvaticus possess higher antibacterial potential against tested Gram-positive than Gram-negative bacterial strains.

The highest activity exhibited hot alkali extract towards E. faecalis. As infections caused by this bacterium are very difficult to treat due to its frequent resistance to multiple antibiotics, the use of A. silvaticus extracts as supplements to certain types of food might lead to the destruction of bacteria in food and thus to contribute to the reduction of poisoning with this type of bacteria.

At a time of increasing resistance of microorganisms to antibiotics, naturally-derived antimicrobial substances are very desirable.

Conclusion

Conclusion

The results from different in vitro assay systems, including the inhibition of lipid peroxidation, the scavenging effects on DPPH radical, the reducing power and the ferrous ions chelating effect, demonstrated that these polysaccharide extracts have effective antioxidant activities. These findings could be important in terms of development of natural, easily accessible sources of antioxidant agents which are able to protect the human body from free radicals and to slow down the progress of many chronic diseases. The results of this study confirm a high biological potential of mushroom A. silvaticus.

Due to its very pleasant taste and nutritional value, antibacterial potential as well as a high content of antioxidant components it could be considered as functional food and might be able to contribute to the reduction of cancer risks.

ACKNOWLEDGEMENTS

This investigation was financially supported by EU Commission project AREA, No. 316004 and by the Ministry of Science and Technological Development of the Republic of Serbia, projects III 46001 and III 46010.